It is often desirable to use fluorescence to detect the presence of a compound. For example, assays (such as immunoassays) can be read by detecting fluorescent energy emitted by a fluorescent tag associated with the compound being detected. Fluorescence tags are relatively easy to use, and they avoid hazards and procedures associated with radioactive tags. Useful applications for fluorescent detection include, without limitation, cell imaging, flow cytometry, immunohistochemistry, and immunoassays. Conventional fluorescent dyes include fluorescein, rhodamine, Texas Red and others.
A frequent problem in fluorescence-based assays is interference due to background fluorescence in the sample or reagents used in the assay. Because this background fluorescence often has a relatively short lifetime and low stokes shift, the use of fluorescent tags with large stokes shift or very long lifetimes (time-resolved fluorescence) allows the detection of smaller amounts of the tag in the presence of large amounts of the background. One method of time-resolved fluorescent tagging involves the use of chelated (using organic chelators) lanthanide metals. 1-6 Lanthanide chelation complexes available today require excitation with ultraviolet light (e.g., often below 340 nm 11-15), requiring complex and relatively expensive light sources, such as a nitrogen laser. Two commercial products based on lanthanide time resolved fluorescence are Perkin Elmer's DELFIA® and LANCE™ products (Perkin Elmer Bioproducts, Boston Mass.). The DELFIA® Eu-labeling reagent consists of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N4-tetraacetic acid (DTTA) chelated with Eu3+. The DTTA group forms a stable complex with europium, and the isocyanate group reacts with a free amino group on the protein to form a stable, covalent thiourea bond. The high water solubility and stability of the chelate, in addition to the mild coupling conditions of the isothiocyanate reaction, make it possible to label antibodies with up to 10-20 Eu/IgG. The LANCE™ product is also an isocyanate (ITC) based chelating product.
The literature reports other chelates that are excitable at longer wavelengths. In some cases, these chelates exhibit relatively poor quantum yields and/or inefficient energy transfer from the chelating compound to the metal ion. For example, Eu (III) Schiff base complexes exhibit relatively low quantum yields when the absorption maximum occurs at longer wavelengths. 16 For some chelates, fluorescence is essentially limited to organic solvents, making them unattractive or impractical for biological applications. Martinus et al described a Eu chelate with Michler's ketone [4,4′-bis(N,N-dimethylamino)benzophenone] (“MK”) with absorption maximum at 414 nm. Again, complex formation occurs in non-coordinating solvents, and water molecules may compete with MK for lanthanide coordination sites. 17
Steemers et al. were able to make Europium and Terbium complexes with a series of calix[4]arenes with excitations extended to at least 350 nm. The reported quantum yields are relatively low and energy transfer is relatively inefficient. It is believed that a significant fraction of the excited species are trapped by molecular oxygen resulting in quenching without contributing to luminescence. 18 Werts et al. disclose complexes of lanthanides with Fluorexon (4′,5′bis[N,Nbis(carboxymethyl)aminomethyl]fluorscein) which can be excited with visible light. 19 However these chelates reportedly have relatively low quantum yields (between 1.7-8.9×10−4) due to non-radiative deactivation.
The anion (compound 1a, below) of the aromatic 1,3-diketone, 2-naphthoyltrifluoroacetone (NTA, compound 1, below) forms a highly fluorescent Eu chelate in aqueous solvent, in the presence of the synergistic agent, tri-n-octyl-phosphene oxide (TOPO).20,21